Therapeutic intervention with highly active antiretroviral therapy (HAART) can lead to suppression of HIV-1 plasma viremia to undetectable levels for 3 or more years. However, adherence to complex drug regimens can prove problematic, and subjects may temporarily discontinue HAART for variable periods. We studied 6 HIV-1–infected individuals who stopped therapy. Off HAART, levels of viremia were suppressed to fewer than 500 copies/mL in 2 subjects for more than 12 and more than 24 months, respectively, and in 1 subject for 4 months on 1 occasion. Three subjects failed to contain plasma viremia. Broad and strong HIV-1–specific immune responses were detected in subjects with prolonged suppression of viral replication. This longitudinal study suggests that containment of HIV-1 replication to low or undetectable levels after discontinuation of HAART is associated with strong virus-specific immune responses. Boosting of HIV-1–specific immune responses should be considered as an adjunctive treatment strategy for HIV-1–infected individuals on HAART.
Gabriel M. Ortiz, Douglas F. Nixon, Alexandra Trkola, James Binley, Xia Jin, Sebastian Bonhoeffer, Peter J. Kuebler, Sean M. Donahoe, Marie-Ange Demoitie, William M. Kakimoto, Tom Ketas, Brian Clas, Jonas J. Heymann, Linqi Zhang, Yunzhen Cao, Arlene Hurley, John P. Moore, David D. Ho, Martin Markowitz
The renal Na+/phosphate (Pi) cotransporter Npt2 is expressed in the brush border membrane (BBM) of proximal tubular cells. We examined the effect of Npt2 gene knockout on age-dependent BBM Na+/Pi cotransport, expression of Na+/Pi cotransporter genes Npt1, Glvr-1, and Ram-1, and the adaptive response to chronic Pi deprivation. Na+/Pi cotransport declines with age in wild-type mice (Npt2+/+), but not in mice homozygous for the disrupted Npt2 allele (Npt2–/–). At all ages, Na+/Pi cotransport in Npt2–/– mice is approximately 15% of that in Npt2+/+ littermates. Only Npt1 mRNA abundance increases with age in Npt2+/+ mice, whereas Npt1, Glvr-1, and Ram-1 mRNAs show an age-dependent increase in Npt2–/– mice. Pi deprivation significantly increases Na+/Pi cotransport, Npt2 protein, and mRNA in Npt2+/+ mice. In contrast, Pi-deprived Npt2–/– mice fail to show the adaptive increase in transport despite exhibiting a fall in serum Pi. We conclude that (a) Npt2 is a major determinant of BBM Na+/Pi cotransport; (b) the age-dependent increase in Npt1, Glvr-1, and Ram-1 mRNAs in Npt2–/– mice is insufficient to compensate for loss of Npt2; and (c) Npt2 is essential for the adaptive BBM Na+/Pi cotransport response to Pi deprivation.
Hannah M. Hoag, Josée Martel, Claude Gauthier, Harriet S. Tenenhouse
Tuberous sclerosis (TSC) is an autosomal dominant genetic disorder in which benign hamartomas develop in multiple organs, caused by mutations in either TSC1 or TSC2. We developed a murine model of Tsc2 disease using a gene targeting approach. Tsc2-null embryos die at embryonic days 9.5–12.5 from hepatic hypoplasia. Tsc2 heterozygotes display 100% incidence of multiple bilateral renal cystadenomas, 50% incidence of liver hemangiomas, and 32% incidence of lung adenomas by 15 months of age. Progression to renal carcinoma, fatal bleeding from the liver hemangiomas, and extremity angiosarcomas all occur at a rate of less than 10%. The renal cystadenomas develop from intercalated cells of the cortical collecting duct and uniformly express gelsolin at high levels, enabling detection of early neoplastic lesions. The tumor expression pattern of the mice is influenced by genetic background, with fewer large renal cystadenomas in the outbred Black Swiss background and more angiosarcomas in 129/SvJae chimeric mice. The slow growth of the tumors in the heterozygote mice matches the limited growth potential of the great majority of TSC hamartomas, and the influence of genetic background on phenotype correlates with the marked variability in expression of TSC seen in patients.
Hiroaki Onda, Andreas Lueck, Peter W. Marks, Henry B. Warren, David J. Kwiatkowski
Guillain-Barré syndrome and its variant, Miller-Fisher syndrome, are acute, postinfectious, autoimmune neuropathies that frequently follow Campylobacter jejuni enteritis. The pathogenesis is believed to involve molecular mimicry between sialylated epitopes on C. jejuni LPSs and neural gangliosides. More than 90% of Miller-Fisher syndrome cases have serum anti-GQ1b and anti-GT1a ganglioside antibodies that may also react with other disialylated gangliosides including GD3 and GD1b. Structural studies on LPS from neuropathy-associated C. jejuni strains have revealed GT1a-like and GD3-like core oligosaccharides. To determine whether this structural mimicry results in pathogenic autoantibodies, we immunized mice with GT1a/GD3-like C. jejuni LPS and then cloned mAb’s that reacted with both the immunizing LPS and GQ1b/GT1a/GD3 gangliosides. Immunohistology demonstrated antibody binding to ganglioside-rich sites including motor nerve terminals. In ex vivo electrophysiological studies of nerve terminal function, application of antibodies either ex vivo or in vivo via passive immunization induced massive quantal release of acetylcholine, followed by neurotransmission block. This effect was complement-dependent and associated with extensive deposits of IgM and C3c at nerve terminals. These data provide strong support for the molecular mimicry hypothesis as a mechanism for the induction of cross-reactive pathogenic anti-ganglioside/LPS antibodies in postinfectious neuropathies.
Carl S. Goodyear, Graham M. O’Hanlon, Jaap J. Plomp, Eric R. Wagner, Ian Morrison, Jean Veitch, Lynne Cochrane, Roland W. M. Bullens, Peter C. Molenaar, Joe Conner, Hugh J. Willison
In vitro, fibroblast growth factor-2 (FGF2) has been implicated in cardiomyocyte growth and reexpression of fetal contractile genes, both markers of hypertrophy. However, its in vivo role in cardiac hypertrophy during pressure overload is not well characterized. Mice with or without FGF2 (Fgf2+/+ and Fgf2–/–, respectively) were subjected to transverse aortic coarctation (AC). Left ventricular (LV) mass and wall thickness were assessed by echocardiography preoperatively and once a week postoperatively for 10 weeks. In vivo LV function during dobutamine stimulation, cardiomyocyte cross-sectional area, and recapitulation of fetal cardiac genes were also measured. AC Fgf2–/– mice develop significantly less hypertrophy (4–24% increase) compared with AC Fgf2+/+ mice (41–52% increase). Cardiomyocyte cross-sectional area is significantly reduced in AC Fgf2–/– mice. Noncoarcted (NC) and AC Fgf2–/– mice have similar β-adrenergic responses, but those of AC Fgf2+/+ mice are blunted. A lack of mitotic growth in both AC Fgf2+/+ and Fgf2–/– hearts indicates a hypertrophic response of cardiomyocytes. Consequently, FGF2 plays a major role in cardiac hypertrophy. Comparison of α- and β-cardiac myosin heavy chain mRNA and protein levels in NC and AC Fgf2+/+ and Fgf2–/– mice indicates that myosin heavy chain composition depends on hemodynamic stress rather than on FGF2 or hypertrophy, and that isoform switching is transcriptionally, not posttranscriptionally, regulated.
Jo El J. Schultz, Sandra A. Witt, Michelle L. Nieman, Peter J. Reiser, Sandra J. Engle, Ming Zhou, Sharon A. Pawlowski, John N. Lorenz, Thomas R. Kimball, Thomas Doetschman
To investigate the function of prostaglandin H synthase-1 and synthase-2 (PGHS-1 and PGHS-2) in the normal lung and in allergic lung responses, we examined allergen-induced pulmonary inflammation and airway hyperresponsiveness in wild-type mice and in PGHS-1–/– and PGHS-2–/– mice. Among nonimmunized saline-exposed groups, we found no significant differences in lung function or histopathology, although PGE2 was dramatically reduced in bronchoalveolar lavage (BAL) fluid from PGHS-1–/– mice, relative to wild-type or PGHS-2–/– mice. After ovalbumin sensitization and challenge, lung inflammatory indices (BAL cells, proteins, IgE, lung histopathology) were significantly greater in PGHS-1–/– mice compared with PGHS-2–/– mice, and both were far greater than in wild-type mice, as illustrated by the ratio of eosinophils in BAL fluid (8:5:1, respectively). Both allergic PGHS-1–/– and PGHS-2–/– mice exhibited decreased baseline respiratory system compliance, whereas only allergic PGHS-1–/– mice showed increased baseline resistance and responsiveness to methacholine. Ovalbumin exposure caused a modest increase in lung PGHS-2 protein and a corresponding increase in BAL fluid PGE2 in wild-type mice. We conclude that (a) PGHS-1 is the predominant enzyme that biosynthesizes PGE2 in the normal mouse lung; (b) PGHS-1 and PGHS-2 products limit allergic lung inflammation and IgE secretion and promote normal lung function; and (c) airway inflammation can be dissociated from the development of airway hyperresponsiveness in PGHS-2–/– mice.
Stephen H. Gavett, Sharon L. Madison, Patricia C. Chulada, Paula E. Scarborough, Wei Qu, James E. Boyle, Howard F. Tiano, Christopher A. Lee, Robert Langenbach, Victor L. Roggli, Darryl C. Zeldin
To determine whether the serine/threonine kinase Akt (also known as protein kinase B) is activated in vivo by insulin administration in humans, and whether impaired activation of Akt could play a role in insulin resistance, we measured the activity and phosphorylation of Akt isoforms in skeletal muscle from 3 groups of subjects: lean, obese nondiabetic, and obese type 2 diabetic. Vastus lateralis biopsies were taken in the basal (overnight fast) and insulin-stimulated (euglycemic clamp) states. Insulin-stimulated glucose disposal was reduced 31% in obese subjects and 63% in diabetic subjects, compared with lean subjects. Glycogen synthase (GS) activity in the basal state was reduced 28% in obese subjects and 49% in diabetic subjects, compared with lean subjects. Insulin-stimulated GS activity was reduced 30% in diabetic subjects. Insulin treatment activated the insulin receptor substrate-1–associated (IRS-1–associated) phosphoinositide 3-kinase (PI 3-kinase) 6.1-fold in lean, 3.7-fold in obese, and 2.4-fold in diabetic subjects. Insulin also stimulated IRS-2–associated PI 3-kinase activity 2.2-fold in lean subjects, but only 1.4-fold in diabetic subjects. Basal activity of Akt1/Akt2 (Akt1/2) and Akt3 was similar in all groups. Insulin increased Akt1/2 activity 1.7- to 2.0-fold, and tended to activate Akt3, in all groups. Insulin-stimulated phosphorylation of Akt1/2 was normal in obese and diabetic subjects. In lean subjects only, insulin-stimulated Akt1/2 activity correlated with glucose disposal rate. Thus, insulin activation of Akt isoforms is normal in muscle of obese nondiabetic and obese diabetic subjects, despite decreases of approximately 50% and 39% in IRS-1– and IRS-2–associated PI 3-kinase activity, respectively, in obese diabetic subjects. It is therefore unlikely that Akt plays a major role in the resistance to insulin action on glucose disposal or GS activation that is observed in muscle of obese type 2 diabetic subjects.
Young-Bum Kim, Svetlana E. Nikoulina, Theodore P. Ciaraldi, Robert R. Henry, Barbara B. Kahn
The pathogenesis of septic shock occurring after Pseudomonas aeruginosa pneumonia was studied in a rabbit model. The airspace instillation of the cytotoxic P. aeruginosa strain PA103 into the rabbit caused a consistent alveolar epithelial injury, progressive bacteremia, and septic shock. The lung instillation of a noncytotoxic, isogenic mutant strain (PA103ΔUT), which is defective for production of type III secreted toxins, did not cause either systemic inflammatory response or septic shock, despite a potent inflammatory response in the lung. The intravenous injection of PA103 did not cause shock or an increase in TNF-α, despite the fact that the animals were bacteremic. The systemic administration of either anti–TNF-α serum or recombinant human IL-10 improved both septic shock and bacteremia in the animals that were instilled with PA103. Radiolabeled TNF-α instilled in the lung significantly leaked into the circulation only in the presence of alveolar epithelial injury. We conclude that injury to the alveolar epithelium allows the release of proinflammatory mediators into the circulation that are primarily responsible for septic shock. Our results demonstrate the importance of compartmentalization of inflammatory mediators in the lung, and the crucial role of bacterial cytotoxins in causing alveolar epithelial damage in the pathogenesis of acute septic shock in P. aeruginosa pneumonia.
Kiyoyasu Kurahashi, Osamu Kajikawa, Teiji Sawa, Maria Ohara, Michael A. Gropper, Dara W. Frank, Thomas R. Martin, Jeanine P. Wiener-Kronish
Excess dietary salt intake induces the activity of the kidney arachidonate epoxygenase and markedly increases the urinary excretion of its metabolites. The epoxyeicosatrienoic acids, products of the kidney P-450 arachidonate epoxygenase, inhibit distal nephron Na+ reabsorption. Nucleic acid hybridization studies demonstrated the expression of P-450s 2C23, 2C24, and 2C11 as the predominant kidney 2C isoforms and the lack of significant dietary salt-dependent transcriptional regulation of these proteins. Recombinant P-450s 2C11, 2C23, and 2C24 catalyze arachidonate metabolism to mixtures of epoxy- and monohydroxylated acids. Whereas the arachidonate 11,12-olefin was the preferred target for epoxidation by P-450 2C23 (57% of total products), P-450s 2C11 and 2C24 epoxidized the 11,12-olefins and 14,15-olefins with nearly equal efficiency. Stereochemical comparisons demonstrated that the regiochemical and enantiofacial selectivity of P-450 2C23 matched that of the kidney microsomal epoxygenase and that excess dietary salt does not alter the regiochemical or stereochemical selectivity of the kidney arachidonate epoxygenase. Inhibition and immunoelectrophoresis experiments using antibodies raised against recombinant P-450s 2C11 and 2C23 demonstrated that P-450 2C23 is the major 2C arachidonic acid epoxygenase in the rat kidney and the renal P-450 isoform regulated by excess dietary salt intake.
Vijaykumar R. Holla, Keiko Makita, Peter G. Zaphiropoulos, Jorge H. Capdevila
IL-12 and IL-18 are IFN-γ–inducing cytokines. In the present study, the role of endogenous IL-18 in the induction of IFN-γ by IL-12 was investigated in mice. In the presence of a specific inhibitor of caspase-1 (also known as IL-1β–converting enzyme, or ICE) IL-12–induced IFN-γ from splenocytes was reduced by 85%. Using splenocytes from ICE-deficient mice, IL-12–induced IFN-γ was reduced by 80%. However, the role of ICE was not through processing and release of IL-1β. Neutralizing anti–IL-18 IgG reduced IL-12–induced IFN-γ in splenocytes by 85%. Splenocytes cultured in vitro spontaneously released IL-18 into the extracellular compartment over time. Extracellular levels of IL-18 significantly correlated with IL-12–induced IFN-γ and were reduced in cells obtained from ICE-deficient mice. In vivo, IL-12 administration increased circulating levels of IL-18 in wild-type mice but not in ICE-deficient mice. Both neutralization of IL-18 and ICE deficiency significantly reduced induction of circulating IFN-γ in mice receiving IL-12. The IL-18 precursor was constitutively expressed in the livers and spleens of untreated mice. Furthermore, administration of IL-12 significantly increased liver-associated IL-18 levels. These data demonstrate that endogenous, ICE-cleaved IL-18 significantly contributes to induction of IFN-γ by IL-12.
Giamila Fantuzzi, David A. Reed, Charles A. Dinarello
Activation of the transcription factor nuclear factor-κB (NFκB) is impaired in T cells from patients with renal cell carcinomas (RCCs). In circulating T cells from a subset of patients with RCCs, the suppression of NFκB binding activity is downstream from the stimulus-induced degradation of the cytoplasmic factor IκBα. Tumor-derived soluble products from cultured RCC explants inhibit NFκB activity in T cells from healthy volunteers, despite a normal level of stimulus-induced IκBα degradation in these cells. The inhibitory agent has several features characteristic of a ganglioside, including sensitivity to neuraminidase but not protease treatment; hydrophobicity; and molecular weight less than 3 kDa. Indeed, we detected gangliosides in supernatants from RCC explants and not from adjacent normal kidney tissue. Gangliosides prepared from RCC supernatants, as well as the purified bovine gangliosides Gm1 and Gd1a, suppressed NFκB binding activity in T cells and reduced expression of the cytokines IL-2 and IFN-γ. Taken together, our findings suggest that tumor-derived gangliosides may blunt antitumor immune responses in patients with RCCs.
Robert G. Uzzo, Patricia Rayman, Vladimir Kolenko, Peter E. Clark, Martha K. Cathcart, Tracy Bloom, Andrew C. Novick, Ronald M. Bukowski, Thomas Hamilton, James H. Finke
In schistosomiasis, chronic parasite egg–induced granuloma formation can lead to tissue destruction and fibrosis, which causes much of the morbidity and mortality associated with this disease. Here we show the importance of IL-13 in the pathogenesis of schistosomiasis, and demonstrate, perhaps for the first time, the therapeutic efficacy of an IL-13 inhibitor, sIL-13Rα2-Fc, in the control of hepatic fibrosis. T-helper type 2 (Th2) cytokines dominate the immune response in mice infected with Schistosoma mansoni, yet the specific contributions of IL-13 and IL-4 to the development of fibrosis were not previously investigated. Our studies demonstrate that both cytokines play redundant roles in granuloma formation, which explains the ability of IL-4–deficient mice to form granulomas around eggs. More importantly, however, these studies demonstrate that IL-13 is the dominant Th2-type cytokine regulating fibrosis. IL-13 stimulated collagen production in fibroblasts, and procollagen I and procollagen III mRNA expression was decreased in sIL-13Rα2-Fc–treated mice. Moreover, the reduction in fibrosis observed in IL-4–deficient mice was much less pronounced than that in sIL-13Rα2-Fc–treated animals. Fibrosis is a major pathological manifestation of a number of allergic, autoimmune, and infectious diseases. Thus, our findings provide evidence that IL-13 inhibitors may be of general therapeutic benefit in preventing damaging tissue fibrosis resulting from Th2-dominated inflammatory responses.
Mónica G. Chiaramonte, Debra D. Donaldson, Allen W. Cheever, Thomas A. Wynn
The pathogenesis of type 2 diabetes involves abnormalities in insulin action, insulin secretion, and endogenous glucose output (EGO). However, the sequence with which these abnormalities develop and their relative contributions to the deterioration in glucose tolerance remain unclear in the absence of a detailed longitudinal study. We measured insulin action, insulin secretion, and EGO longitudinally in 17 Pima Indians, in whom glucose tolerance deteriorated from normal (NGT) to impaired (IGT) to diabetic over 5.1 ± 1.4 years. Transition from NGT to IGT was associated with an increase in body weight, a decline in insulin-stimulated glucose disposal, and a decline in the acute insulin secretory response (AIR) to intravenous glucose, but no change in EGO. Progression from IGT to diabetes was accompanied by a further increase in body weight, further decreases in insulin-stimulated glucose disposal and AIR, and an increase in basal EGO. Thirty-one subjects who retained NGT over a similar period also gained weight, but their AIR increased with decreasing insulin-stimulated glucose disposal. Thus, defects in insulin secretion and insulin action occur early in the pathogenesis of diabetes. Intervention to prevent diabetes should target both abnormalities.
Christian Weyer, Clifton Bogardus, David M. Mott, Richard E. Pratley
The differing tempo and direction of growth of the periosteal and endocortical surfaces, and the differing tempo of growth of the axial and appendicular skeleton, may predispose to regional deficits in bone size, bone mineral content (BMC), and volumetric bone mineral density (vBMD). These traits were measured during 2 years by dual x-ray absorptiometry in 109 girls. By 7 years of age, bone size was approximately 80% of its maturational peak, and BMC was approximately 40% of its peak. Before puberty, the legs grew more rapidly than the trunk. During puberty, the growth spurt was truncal. Between 7 and 17 years, femoral and lumbar spine BMC increased by 50–150% because bone size increased. vBMD increased by 10–30%. Thus, growth builds a bigger, but only moderately denser, skeleton. Regions growing rapidly, or distant from their peak, may be more severely affected by illness than those growing slowly or nearer completion of growth. Depending on the age of exposure to disease, deficits may occur in limb dimensions (prepuberty), spine dimensions (early puberty), or vBMD by interference with mineral accrual (late puberty). As vBMD is independent of age before puberty, the position of an individual’s vBMD in the population distribution is established early in life. Bone fragility in old age may have its foundations in growth.
Shona Bass, Pierre D. Delmas, Georgina Pearce, Elke Hendrich, Aaron Tabensky, Ego Seeman
To address the hypothesis that elevated blood alcohol increases systemic oxidant stress, we measured urinary excretion of isoprostanes (iPs), free radical–catalyzed products of arachidonic acid. Ten healthy volunteers received acute doses of alcohol (Everclear-R) or placebo under randomized, controlled, double-blind conditions. Urinary iPF2a-III increased in a time- and dosage-dependent manner after dosing with alcohol, with the peak urinary iPF2a-III excretion correlating with the rise in blood alcohol. To determine whether oxidant stress was associated with alcohol-induced liver disease (ALD), we then studied the excretion of iP in individuals with a documented history of alcohol-induced hepatitis or alcohol-induced chronic liver disease (AC). Both urinary iPF2a-III and urinary iPF2a-VI were markedly increased in patients with acute alcoholic hepatitis. In general, urinary iPF2a-III was significantly elevated in cirrhotic patients, relative to controls, but excretion was more pronounced when cirrhosis was induced by alcohol than by hepatitis C. Excretion of iPF2a-VI, as well as 4-hydroxynonenal and the iPF2a-III metabolite, 2,3-dinor-5,6-dihydro-iPF2a-III, was also increased in AC. Vitamin C, but not aspirin, reduced urinary iPs in AC. Thus, vasoactive iPs, which serve as indices of oxidant stress, are elevated in the urine in both acute and chronic ALD. Increased generation of iPs by alcohol in healthy volunteers is consistent with the hypothesis that oxidant stress precedes and contributes to the evolution of ALD.
Emma A. Meagher, Orla P. Barry, Anne Burke, Michael R. Lucey, John A. Lawson, Joshua Rokach, Garret A. FitzGerald
Angiotensin II (Ang II) has been shown to stimulate either hypertrophy or hyperplasia. We postulated that the differential response of vascular smooth muscle cells (VSMCs) to Ang II is mediated by the cyclin-dependent kinase (Cdk) inhibitor p27Kip1, which is abundant in quiescent cells and drops after serum stimulation. Ang II treatment (100 nM) of quiescent VSMCs led to upregulation of the cell-cycle regulatory proteins cyclin D1, Cdk2, proliferating cell nuclear antigen, and Cdk1. p27Kip1 levels, however, remained high, and the activation of the G1-phase Cdk2 was inhibited as the cells underwent hypertrophy. Overexpression of p27Kip1 cDNA inhibited serum-stimulated [3H]thymidine incorporation compared with control-transfected cells. This cell-cycle inhibition was associated with cellular hypertrophy, as reflected by an increase in the [3H]leucine/[3H]thymidine incorporation ratio and by an increase in forward-angle light scatter during flow cytometry at 48 hours after transfection. The role of p27Kip1 in modulating the hypertrophic response of VSMCs to Ang II was further tested by antisense oligodeoxynucleotide (ODN) inhibition of p27Kip1 expression. Ang II stimulated an increase in [3H]thymidine incorporation and the percentage of S-phase cells in antisense ODN–transfected cells but not in control ODN–transfected cells. We conclude that p27Kip1 plays a role in mediating VSMC hypertrophy. Ang II stimulation of quiescent cells in which p27Kip1 levels are high results in hypertrophy but promotes hyperplasia when levels of p27Kip1 are low, as in the presence of other growth factors.
Ruediger C. Braun-Dullaeus, Michael J. Mann, Almut Ziegler, Heiko E. von der Leyen, Victor J. Dzau
B. Lawrence Riggs, Sundeep Khosla, L. Joseph Melton III
Kenneth Walsh, Ichiro Shiojima, Antonio Gualberto
Matthew J. Brady, Alan R. Saltiel